Significance Contrast ultrasound involves the injection of echo-producing, biologically compatible solutions or particles into the bloodstream to enhance the echogenicity of a structure. Clinical applications of contrast media include the determination and evaluation of normal and abnormal cardiac structures, assistance in the diagnosis of shunts and valvular regurgitations, and visualization of blood flow. In order for the material to be effective it must be stable throughout the examination, but metabolized or removed safely from the circulation shortly thereafter and must also have preferential biodistribution and tissue uptake with relatively low toxicity, such that sufficient quantities can be administered. Objectives This study was designed to addressed issues such as safety, stability, uniformity, reproducibility, and echogenicity with no effects on the circulation of a new echocontrast agent proposed for use in human patients. Results Harmonic and real-time and triggered imaging was performed before, during, and after injections of microspheres into 5 rhesus monkeys at differing doses. Results showed prolonged endocardial enhancement with full left ventricular opacification lasting 30 minutes or more in parasternal and apical views. Contrast enhancement of both liver and kidney was also evident, with liver opacification gradually increasing over the study period. Heart rate, blood pressure, oxygen saturation, electrocardiograms, and blood tests (hematology, clinical chemistries, coagulation panels, and CPK-MB) remained stable during and following injections. Initial and three-day follow-up studies showed no apparent clinical or significant abnormalities. Thus, prolonged cardiac, liver, and kidney contrast opacification was safely achieved suggesting that diffusible gases can produce safe, long-lasting echocontrast agents without the need for fluorocarbon gases. Future Directions Studies with rhesus monkeys will continue to explore safe and effective echocontrast agents for use in the human clinical setting. KEY WORDS echocontrast agents, monkey, safety, efficacy, ultrasound FUNDING Private Sector